The main objective of this research program is to obtain detailed information about the mechanism by which certain anticancer drugs such as methotrexate exert their inhibitory action on the biological target site dihydrofolate reductase. Although research in the past decade has revealed the major steps in the reaction pathway of this key enzyme, i.e., the substrate has the 7,8-dihydro structure and hydrogen is transferred stereospecifically from the A-side of TPNH to carbon 6 of the substrate without rearrangement giving the active coenzyme tetrahydrofolate, relatively little has been learned about the mechanism involved in either the reaction or its inhibition. Such information would provide a more rational basis for design of new antifolate chemotherapeutic agents. Our initial results obtained by proton magnetic resonance (PMR) spectroscopy reveal the efficacy and sensitivity of this method in observing interaction of the enzyme with substrates, cofactors and inhibitors. Small structural differences of the ligand are reflected in the enzyme PMR spectrum providing evidence of conformation response and thereby the means of possibly identifying these changes in terms of the specific amino acid residues involved. By applying several recent methodological advances, we are optimistic that these objectives will be attained. Procedures and results developed studying this more readily available enzyme will increase our efficiency in extracting data from more refractory but important mammalian enzyme systems.